PT1514482E - Milk fractions and milk preparations for treating and/or preventing cox-2 mediated diseases - Google Patents

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF MILK AND MILK PREPARATIONS FOR TREATMENT AND / OR PREVENTION OF COX-2 MEDIATED DISEASES " The present invention relates to pharmaceutical and nutritional compositions for the treatment and / or prevention of cyclooxygenase-2 mediated diseases and to a process for enhancing the cyclooxygenase-2 inhibitory activity of a product. In particular, the present invention also relates to the use of one or more milk protein fractions and / or one or more milk protein preparations for the inhibition of cyclooxygenase-2 activity, specifically, for the preparation of a food product or a medicament for the treatment and / or prevention of cyclooxygenase-2 mediated diseases.

The cycloxigenase (COX) enzymes are key enzymes necessary for the conversion of arachidonic acid to prostaglandins (PG). So far, two COX, COX-1 and COX-2 isoforms have been identified, which differ in several respects: COX-1 is constitutively expressed in most tissues and is presumed to be responsible for the production of prostaglandins that control functions physiological conditions, such as maintenance of the gastric mucosa and regulation of renal blood flow. The second isoform, COX-2, does not appear to be constitutively expressed in most normal tissues, but is highly inducible and can be found only in particular locations, e.g. in inflammation sites and in cancer cells. 1

Although it is found that the COX enzymes are membrane bound, they do not have a conventional transmembrane region. Instead, they contain four overlapping amphipathic helices to form a region of hydrophobicity. This hydrophobic region attaches to the " lower " of the enzyme in the membrane. The cyclooxygenase active site is located in the area of hydrophobicity near the amphipathic helices. The substrate and enzyme inhibitors are believed to reach the active site via a channel included in the lipid bilayer (W. Krause, R.N. Dubois, Prostaglandins & other Lipid Mediators 61 (2000), 145-161).

COX inhibitors, and in particular COX-2 inhibitors, are used to treat a variety of different diseases, such as inflammatory diseases and are also used as analgesics. In addition, COX-2 has been found to be normally overexpressed in pre-malignant and malignant tissues, and a treatment with COX-2 inhibitors has been shown to reduce the formation of intestinal, esophageal, tongue, breast, skin tumors , lung and bladder in animals (K. Subbaramaiah, AJ Dannenberg, TRENDS in Pharmacological Sciences 24 (2003), 96-102). Various well known non-steroidal anti-inflammatory drugs (designated by NSAIDs), such as e.g. aspirin and ibuprofen, inhibit COX-1 and COX-2. Recently, a new class of inhibitors (COXIB) has been described, which only specifically inhibits the COX-2 enzyme (Μ E. Turini, RN Dubois, Physiology And Diseases, Annu Rev. Med. 35-57). However, NSAIDs are known to cause serious side effects, e.g. kidney problems and duodenal and stomach ulceration. WO 01/11990 discloses pharmaceutical and nutritional compositions with reduced threonine content. Said compositions may comprise, as a major ingredient, whey acid whey protein or sweet whey protein from which casein glycocro-peptide was removed. Removal of said casein protein is achieved by contacting sweet whey protein concentrate with a cation exchange resin followed by treatment with a weakly anionic resin. A combination preparation directed to a decreased risk of civilizational diseases is described in DE 4413839. Said preparation comprises, inter alia, sweet whey protein, which is described as interacting with other constituents, magnesium, vitamin C, vitamin E, provitamin A, vitamin B1 and selenium in order to provide the desired therapeutic effect. US 4284623 discloses a method for treating inflammation in an animal, which comprises administering to the animal an anti-inflammatory effective amount of milk of a bovine maintained in an anti-inflammatory factor producing state.

Accordingly, the problem of the present invention is to provide additional means for inhibiting cyclooxygenase, in particular, COX-2, the means of which must be associated with a low risk of deleterious side effects.

This problem has been solved by providing specific fractions and / or milk preparation, which exhibit COX-2 inhibitory activity, for the treatment and / or prevention of COX-2 mediated diseases. 3

In the figures, Fig. 1 is a diagram showing the preparation of bovine milk fractions, as shown in Example 1.1; Fig. 2 is a diagram showing the preparation of human milk fractions as shown in Example 1.2; Fig. 3 is a diagram showing the preparation of buffalo milk fractions, as shown in Example 1.3; and Fig. 4 shows a comparison of the COX-2 inhibitory activities of various milk protein fractions and milk protein preparations derived from human milk (HM), cow's milk (CM) and buffalo milk ( BM).

According to one aspect, the present invention provides the use according to claim 1. The term " treating and / or preventing cyclooxygenase-2 mediated diseases " as used in the present application comprises treating a disease associated with expression in particular with an increased expression of cyclooxygenase-2 and to prevent a disease, which may be influenced by reduction of the level of cyclooxygenase-2 at a specific site in the body of an individual, or by preventing the increase in the level of cyclooxygenase- 2, at a specific site, in the body of an individual. An increased level of cyclooxygenase-2 designates an amount, which is higher than the level of cyclooxygenase-2 statistically present, on average, in a healthy individual at a specific site. 4

A first group of cyclooxygenase-2 mediated diseases, which can be treated and / or their prevented onset, according to the present invention, is cancer, comprising any of its pre-cancerous conditions. According to available scientific data, presently, it is presumed that in pre-malignant and malignant tissues, COX-2 is overexpressed and that said increased COX-2 expression is associated, at least in part, with induction of tumor formation. In fact, such overexpression of COX-2 has been observed in various pre-malignant and malignant conditions in organs, such as, e.g. colon, stomach, esophagus, liver, bile, pancreas, head and neck, lung, breast, bladder, female genital organs and skin. In addition, successful treatment of tumors based on prior art COX-2 inhibitors has also been achieved. Thus, administration of a composition according to the present invention will be useful for treating or preventing cancerous and / or precancerous conditions.

In addition, COX-2 inhibitors have been shown to block the formation of prostaglandins, the presence of which is associated with the development and progression of pain, fever and inflammation. According to the present invention, other target groups for cyclooxygenase-2 mediated diseases for which a treatment and / or prevention is directed are arthritis, rheumatic fever, symptoms associated with influenza or other viral infections, dysmenorrhea, headache , toothache, degenerative joint diseases, etc. Alzheimer's disease is also considered to be a target disease in accordance with the present invention since COX-2 inhibitors have been shown to have a protective effect in the development of this disease. The use of the present invention is designed for any individual in need thereof, preferably mammals, in particular, humans and animals, e.g. g.r pets.

A " cyclooxygenase-2 inhibitory activity " as mentioned herein may be determined according to a HUV-EC-C cell based COX-2 screening method, as indicated in the examples below, but also based in other assays known in the art. The therapeutically effective amount of one or more milk protein fractions and / or one or more milk protein preparations specifically required for a given subject can be readily determined by one of ordinary skill in the art, according to their general knowledge in the art, considering a variety of factors, such as body weight, age, overall health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, etc. Basically, the specific dosage regimen for a particular subject will depend on whether the target is either a general prevention or an acute treatment of a disease. For example, the daily dosage of said one or more milk protein fractions and / or one or more milk protein preparations may be selected from 7 mg to 70 g in the case of a 70 kg person, corresponding to a daily dosage of from about 0.1 mg to about 1 g per kilogram of body weight, preferably from about 0.5 mg to about 100 mg, more preferably from about 5 mg to about 70 mg, and even more preferably from about 10 mg to about 50 mg. The nutritional composition prepared by the use of the present invention is any composition suitable for human or animal consumption comprising at least one material selected from the group consisting of water, proteins and / or peptides, carbohydrates, fats. The pharmaceutical composition prepared by the use of the present invention is any composition comprising at least one therapeutically active compound as set forth herein.

In addition, the present invention provides a nutritional and / or pharmaceutical composition, prepared by the use of the present invention, which comprises an increased amount of said one or more milk protein fractions and / or one or more milk protein preparations providing a cyclooxygenase-2 inhibitory activity and which provides a cyclooxygenase-2 inhibitory activity that is superior to the cyclooxygenase-2 inhibitory activity of a nutritional and / or pharmaceutical composition, wherein the amount of said one or more protein fractions of milk and / or one or more milk protein preparations was not increased.

Such an increased amount of said one or more milk protein fractions and / or one or more milk protein preparations providing a cyclooxygenase-2 inhibitory activity may be obtained by adding an additional amount of said one or more fractions of milk protein and / or one or more milk protein preparations and / or by substituting a milk fraction with a milk fraction in which the bioactive compound (s) has been (en) enriched ( s). An enrichment can be achieved according to physical, chemical and biological techniques well known in the art and can be carried out e.g. based on the assay described in the examples. The cyclooxygenase-2 inhibitory activity of a product resulting from an enrichment or addition, as set forth above, should be, for example, at least 1%, more preferably at least 5%, even more preferred , at least 10% higher, and in particular at least 25% higher, to the cyclooxygenase-2 inhibitory activity of the reference nutritional / pharmaceutical composition, i. of the nutritional / pharmaceutical composition having - in addition to one or more milk protein fractions and / or one or more additional and / or enriched milk protein preparations - the same composition as the nutritional / pharmaceutical composition having an inhibitory activity of cyclooxygenase-2.

During extensive studies, the present inventors have demonstrated that specific fractions of milk protein provide a cyclooxygenase-2 inhibitory activity, which essentially may not be found at or exceeds that of milk as such. Without wishing to be bound by any theory, it may be assumed that the low initial activity observed in human milk appears to be associated with whey proteins. The caseins themselves may have a suppressive effect or contain " latent bioactivity " which is activated (and then found in the whey fraction) by acidification or rennet treatment.

As it is found that said milk fractions exhibit increased COX-2 inhibitory activity, there may be mentioned fractions of sweet whey of human milk, bovine milk, sweet bovine milk casein fraction, etc. or combinations thereof. Further sub-fractionation of soluble whey protein and screening reveals that some sub-fractions of soluble whey protein from bovine milk are preferred.

In principle, the separation and isolation of soluble whey protein sub-fractions can be achieved by hydrophobic interaction chromatography (HIC), or by hydrophobic interaction high pressure liquid chromatography (HI-HPLC), high pressure liquid chromatography (RP-HPLC), and the like, all of which are based on the same principles of separation, for example. The principles of HIC are known to the person skilled in the art. Generally, samples are loaded onto a balanced column (stationary phase) comprising a hydrophobic material that retains the samples. The hydrophobic material may be, for example, macroporous crosslinked polystyrene, available as Amberlite Xad 16 XAD 16 from Rohm and Hass, for example. Amersham's 15 RPC TN 17-0727-02 (polystyrene-divinylbenzene) or the like may also be used.

Before the protein is loaded onto the column, it can be equilibrated with a buffer. After the fraction is loaded, the run can be run through the column of a buffer or a mixture of buffers (mobile phase) whereby the buffer mixture varies and may thus have varying properties of eluent protein sub-fractions , according to their hydrophobicity from the column. Separation of whey proteins according to this method is described in: " Simultaneous separation and quantitation of the major bovine whey proteins including protease peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene " , DF Elgar et al., Journal of Chromatography A 878 (2000) 183-196.

The protein sub-fractions eluted from the column can be accurately described by the composition of the buffer mixture or acetonitrile content which eluted from the stationary phase. For example, the soluble whey protein can be loaded onto a column filled with polystyrene-divinylbenzene beads (15 RPC TN 17-0727-02 from Amersham) a buffer A can be defined as 0.1% vol trifluoroacetic acid (TFA ) in water and a buffer B may be defined as 80% vol of acetonitrile and 0.85% vol of TFA (% vol). Thereafter, mixing and transporting the buffers A and B can be controlled by a specific system, for example, a UNICORN (Pharmacia / Amersham) FPLC (Fast Protein Liquid Chromatography) station and flowed through the column. The eluted protein sub-fraction may be defined by a range of elution ratios of mixing the above-mentioned buffers A and B. Using the specific buffer composition (because the elution order depends on pH), the elution or interval time of a protein sub-fraction can be simply described by the relative amount of acetonitrile present at the time of elution of a protein fraction of according to the invention.

If desired, the sub-fractions thus obtained may be concentrated by techniques well known in the art, such as evaporation, ultrafiltration or dialysis to remove organic solvent prior to drying, for example by vacuum, freezing, spraying, fluidized bed, furnace, or any other suitable drying process. 10

The sub-fractions shown in the examples in Table 7 have been shown to be particularly effective in promoting inhibition of COX II. According to an even more preferred embodiment, the present invention relates to sub-fractions 1, 9, 10 and 14, as shown in Table 7, which are highly effective.

Generally, human milk, cows, can be used to prepare the milk protein fractions or protein preparations according to the present invention.

Lean milk, acid whey and sweet whey may be prepared according to a process as described in the examples. The terms " soluble whey protein " or " soluble protein ", as used in the context of the present invention, means protein recovered in aqueous solution after ultra-centrifugation (e.g., for 1 h at 100,000 g) or according to another method known to a person skilled in the art. The terms " non-soluble protein " or " micellar casein " indicate the washed material recovered from the pellet after said ultracentrifugation (e.g., according to the steps as set forth in example 1 or according to another method known to a person skilled in the art).

Particularly, high inhibitory activities of cyclooxygenase-2 are provided by sweet whey of human milk and by various fractions of cow's milk, i. sweet whey, sweet casein. For all three milk species investigated in the examples (human, bovine, buffalo), the highest higher COX-2 inhibition rates are obtained with sweet whey which accordingly represent a preferred embodiment of the present invention. 11

Whey protein preparations provided good results, however, they were not as effective as several fractions of milk protein. Thus, it may be assumed that in the milk protein fractions according to the present invention, positive synergistic effects are provided which increase the COX-2 inhibitory activity. If desired, the milk protein fractions and the whey protein preparations may also be combined.

Studies directed to the identification of the cyclooxygenase-2 inhibiting compound (s) present in the milk protein fractions according to the present invention have shown that the compounds must be proteinaceous. Lactoferrin, a known compound, has been shown to have no cyclooxygenase-2 inhibitory activity. The bioavailability and stability with respect to the biodegradation of said one or more milk protein fractions and / or one or more milk protein preparations may further be enhanced by protecting one or more functional groups, such as e.g. -OH, -NH 2, -SH, -COOH, of compounds present in said one or more milk protein fractions and / or one or more milk protein preparations. Said protection can be performed, e.g. by a reaction with one or more sugars or carbohydrates, such as e.g. lactose. For example, heating of milk proteins in the presence of lactose provides lactosylated proteins. After protection, the compounds will provide increased resistance against degradation, in particular, a proteolytic degradation in the body which, in turn, generally results in increased bioavailability. A derivatization may also be used to obtain a desired solubility and thus a desired and controlled bioavailability. The present invention relates to the use of claim 1. One or more milk protein fractions and / or one or more milk protein preparations providing COX-2 inhibitory activity may be administered, e.g. in an oral, topical, parenteral or rectal dosage form containing conventional pharmaceutically acceptable carriers, adjuvants and excipients and conventional non-toxic carriers. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques. The pharmaceutical or nutritional composition according to the present invention may be used to treat or prevent cyclooxygenase-2 mediated diseases, such as cancer or precancerous conditions, in particular, of the colon, stomach, esophagus, liver, biliary body , pancreas, head and neck, lung, breast, bladder, female genitals and skin and / or inflammatory diseases such as arthritis, rheumatic fever, symptoms associated with influenza or other viral infections, dysmenorrhea, headache, toothache, degenerative diseases of articulation, etc. and / or Alzheimer's disease.

In addition, the nutritional or pharmaceutical composition according to the present invention may be applied as a co-treatment during a conventional cancer treatment and / or a pre-cancerous treatment, chemotherapy, radiation therapy, a biotherapy and / or during the treatment of an inflammatory disease and / or during the treatment of Alzheimer's disease. The one or more milk protein fractions and / or one or more milk protein preparations providing a cyclooxygenase-2 inhibitory activity may also be useful as a partial or complete substitute for conventional medicaments, such as e.g. g-. NSAID. Thus, the invention comprises pharmaceutical compositions for treating cyclooxygenase-2 mediated diseases as defined above comprising a therapeutically effective amount of said one or more milk protein fractions and / or one or more milk protein preparations and one or more therapeutically effective ingredients, such as an analgesic; an enhancer; an H2 antagonist, a decongestant; an antitussive; a diuretic; a sedative or non-sedative antihistamine; an agent for treating or preventing cancer and / or precancerous conditions, etc.

The pharmaceutical compositions containing the active ingredient (s) may be in any form suitable for oral use, such as e.g. tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

Compositions designed for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservatives so as to provide pharmaceutically elegant and tasty preparations. 14

The tablets contain the active ingredient (s) in admixture with non-toxic pharmaceutically acceptable excipients, such as diluents, granulants, disintegrating agents and inert lubricants, which are suitable for the manufacture of tablets. The tablets may be uncoated or may be coated by known techniques to retard disintegration and absorption in the gastrointestinal tract and thus provide sustained action over a longer period.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent or as soft gelatin capsules wherein the active ingredient is mixed with water or an oily medium.

The aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions, such as e.g. suspending agents, dispersing or wetting agents, preservatives, coloring agents, flavoring agents and sweetening agents.

Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient (s) in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives . Other excipients, for example, sweetening, flavoring and coloring agents, may also be present.

Milk protein fractions and / or milk protein preparations, as illustrated above, may be used in any functional nutritional, pharmaceutical or food composition, the compositions of which may comprise, e.g. at least one component of the group consisting of water, proteins and / or peptides, fats and carbohydrates. Said composition may further comprise minerals and vitamins, e.g. in an amount of the form 30% to 150% of the daily dosage according to U.S. RDA. In addition, such compositions may comprise fiber material, flavoring agents, emulsifiers, radical traps, preservatives, acids, lipids, fruit and fruit juices, teas, etc.

In particular, the compositions prepared by the use of the present invention may comprise, e.g. a food composition selected from milk, yogurt, curds, cheese, fermented milks, fermented milk products, ice creams, cereal based products, milk based powders, infant formulas or pet food.

Formulations which are expected to be widely accepted by consumers are, for example, beverages, e.g. based on dairy products, conventional beverages, such as water, fruit juices or teas, comprising the milk protein fractions and / or preparations prepared by the use of the present invention.

Further examples for advantageous formulations are e.g. compositions in the form of dessert preparations, gels or foams (such as, eg, mousses " foams "), white delicacies, gums, flans, yogurt-based preparations) or in the form of meals comprising a bakery product having a filler or center comprising the milk protein fractions and / or preparations according to the present invention. Milk protein fractions and / or preparations prepared by the use of the present invention may also be applied, e.g. in the form of a top layer or coating in any conventional food product or be included as a filler in any conventional food product. The product prepared by the use of the present invention, wherein the cyclooxygenase-2 inhibitory activity is increased, may be e.g. a nutritional and / or a pharmaceutical and / or a functional food composition, in particular a dairy food product. The process for isolating sweet whey protein providing a cyclooxygenase-2 inhibitory activity comprises the following steps: centrifuging milk at 13600 g for 30 minutes to obtain a cream layer and a thin milk layer; separating said layer of cream; isolating said lean milk; adding an aqueous CaCl 2 solution to the skim milk in an amount such that a mixture having a concentration of 2 mmol / L Ca 2+ is obtained; heating said mixture to 35øC and adding rennet having an activity of 50 mg of active chymosin per liter (eg, in an amount of 0.4 to 0.6, preferably 0.5 pL rennet per ml of lean milk) into said mixture or heating said mixture to 25 ° C and acidifying said mixture to a pH of 4.6 with an aqueous solution of HCl (32%); centrifuging the mixture at 13600 g for 30 minutes to obtain phase separation; and isolating the whey protein sweet. The process for isolating soluble whey protein providing a cyclooxygenase-2 inhibitory activity comprises the following steps: centrifuging milk at 13600 g for 30 minutes to obtain a cream layer and a thin milk layer; separating said layer of cream; isolating said lean milk; adding an aqueous CaCl 2 solution to the skim milk in an amount such that a mixture having a concentration of 2 mmol / L Ca 2+ is obtained; centrifuging for 1 hour at 100,000 g to separate the soluble whey protein phase from the non-soluble micellar casein phase; and isolating the soluble whey protein phase.

Because of its cyclooxygenase-2 inhibitory activity, the milk protein fraction prepared by the use of the present invention will provide alternatives to conventional NSAIDs, in particular, in cases where said NSAIDs are contraindicated, such as in the case of individuals who suffer from ulcers, coagulation disorders, kidney diseases, etc. Furthermore, it can be expected that such a product will be essentially free of harmful side effects and thus can be consumed even over a long period of time. This will be particularly of high interest when a prevention of a cyclooxygenase-2 mediated disease is desired, e.g. in the case of persons who are presumed to have an increased risk of developing cancer because of their genetic predisposition or because of exposure to well-known risk factors (high-risk nutrition such as, eg, a typical "; Western food "high in fats and low in fiber, exposure to chemicals, etc.).

Since milk products have long been consumed by man, it can be expected that a functional food or medicament prepared by the use of the present invention will be very well accepted by consumers. 18

The following examples illustrate the invention without limiting it to the specific embodiments mentioned.

Examples

All solvents were analytical grade or HPLC and were purchased from Merck (Dietikon, Switzerland). Water was purified internally using a Millipore Milli-Q water purification system (Millipore, Volketswil, Switzerland) or was HPLC grade (Merck, Darmstadt, Germany). CaCl 2 x 2 H 2 O, HCl (32%), sodium hydroxide, peridrol 30% H2O2. A. They were all from Merck.

(&Quot; simple pressure ") was produced by TEXEL (38470 Vinay, France) and was obtained from Rhone Poulenc Rohrer (Cooperation Pharmaceutique Francaise, 77000 Melun, France; Lot No. 101089007) with an activity of 50 mg chymosin active per liter.

Example 1

Preparation of Milk Sample

Standard steps for the preparation of milk fractions were performed as described in: C. Alais, Science du Lait, Prince of Techniques Laitieres. SEPAIC, Paris, 4th ed., 1984, p. 29-35 and 159-178, unless otherwise explicitly indicated.

The indicated laboratory scale fractionation steps have been developed from conventional industrial milk processes. Surprisingly, the selectivity and separation efficacy could be significantly increased by performing the process steps listed below. In particular, by performing a centrifugation at high acceleration rates and specifically tailored washing steps. 1.1 Bovine milk Bovine milk was obtained from a local market (" Toni lait ", Switzerland).

In a first step, the cream was extracted from whole milk by means of a centrifugation having an acceleration rate up to 13600 g using a Sorvai GS3 fixed angle rotor (9000 rpm, for 30 minutes). Starting from 2200 ml of whole milk, 90 g of cream were recovered in the top layer. After separation of the upper layer, 2090 mL of lean milk was obtained. The separation of sweet whey from casein is achieved by enzymatic treatment of lean milk inducing casein coagulation. 520 μl of a 200 mM CaCl 2 aqueous solution (200 mmol / L) was added to 520 ml of skim milk in order to achieve a mixture having a final concentration of 2 mM (mmol / L) Ca 2+. This lean milk mixture was heated to 35Â ° C and then 250 Âμl rennet were immediately added under moderate magnetic stirring. After 1 min, the mixture was incubated for 50 min (minute (s)) at 35øC in a water bath, poured into bottles for centrifugation and subsequently centrifuged for 30 minutes at 13600 g 20 (fixed-angle rotor Sorvai GS3; 9000 rpm, 30 min) in order to separate sweet whey from casein from non-soluble rennet.

The 766 mL supernatant (i.e., sweet whey) was fractionated into aliquots of 10 x 1.3 mL (Eppendorf) and 40 mL plastic tubes, which were labeled (Whey sweet), frozen by immersing in liquid nitrogen and stored in a plastic bag at minus 20 ° C. The rennet casein (45 g) was dispersed in 286 ml of a 2 mM CaCl 2 aqueous solution (mmol / L), said CaCl 2 solution having been supplemented with 0.9% by weight NaCl and then, centrifuged; the supernatant (246 mL) was divided into reservoirs, which were labeled (rennet casein wash) and frozen in liquid nitrogen.

The 31 g of washed rennet casein recovered were dispersed in a 2 mM CaCl 2 aqueous solution (mmol / L), said CaCl 2 solution having been supplemented with 0.9% by weight NaCl, to give volume of 250 mL. The mixture was then divided into reservoirs, which were labeled (rennet casein) and frozen. The separation of lactic acid whey from casein is obtained by chemical acidification of lean milk inducing casein coagulation. 520 μl of a 200 mM (mmol / L) aqueous solution of CaCl 2 was added to 520 ml of skim milk to provide a mixture having a final concentration of 2 mM (mmol / L) Ca 2+. This mixture was acidified to 25 ° C by addition of an aqueous solution of HCl (32%) from pH 6.6 to pH 4.6 under moderate magnetic stirring. After 1 min of stirring, the mixture was incubated for 60 min at 25øC, poured into bottles for centrifugation and subsequently centrifuged for 30 min at 13600 g (Sorvai GS3 fixed angle rotor 9000 rpm for 30 min) to separate whey acid from non-soluble acid casein.

The 503 mL of supernatant (ie, acid whey) were fractionated in 10 x 1.3 mL aliquots (Eppendorf) and 40 mL plastic tubes, which were labeled (acidic whey) and frozen by soaking in nitrogen and stored in a plastic bag at -20 ° C. Acid casein (41 g) was dispersed in 233 ml of a 20 mM (mmol / L) aqueous sodium acetate solution having pH 4.6. The thus obtained mixture was centrifuged and the supernatant (250 mL) was separated and subsequently partitioned into reservoirs, which were labeled (acidic casein wash) and frozen in liquid nitrogen.

The 28.6 g recovered from washed acid casein were dispersed in water. The pH of this mixture was adjusted from 4.67 to 6.6 by an addition of NaOH. Subsequently, the volume was adjusted to 250 mL and the mixture thus obtained was divided into reservoirs, which were labeled (Acid Casein) and frozen.

In order to separate soluble proteins (whey) and washed non-soluble proteins (micellar casein), the following steps were performed: 250 μl of a 200 mM aqueous CaCl 2 solution (mmol / L) was added to 250 ml of skim milk to obtain a mixture having a final concentration of 2 mM (mmol / L) Ca 2+. This milk 22 was ultra-centrifuged (6 tubes containing 42.1 g of skim milk in a fixed-angle rotor) and centrifuged for 1 h in a Beckman L8-60M ultracentrifuge at a speed of 32000 rpm corresponding to 100000 g in the middle of the tube ) in order to separate soluble whey protein from non-soluble micellar casein.

The 228 mL supernatant (ie, soluble whey protein) was fractionated in 10 x 1.3 mL aliquots (Eppendorf) and 40 mL plastic tubes, which were labeled (soluble whey protein) and frozen by immersion in liquid nitrogen and stored in a plastic bag at minus 20 ° C. Micellar casein (24 g) was dispersed in 220 mL of a 2 mM aqueous CaCl 2 solution (mmol / L), said CaCl 2 solution being supplemented with 0.9% by weight NaCl. The mixture was ultra-centrifuged (in a Beckman L8-60M ultracentrifuge for a speed of 32,000 rpm corresponding to 100,000 g in the middle of the tube. The supernatant (229 mL) was separated and then partitioned into reservoirs, which were labeled (Micellar casein washing) and frozen in liquid nitrogen.

The 22 g of washed micellar casein were dispersed in a 2 mM CaCl 2 aqueous solution (mmol / L), said CaCl 2 solution being supplemented with 0.9% by weight NaCl, to give a volume end of 250 mL. This mixture was divided into reservoirs, which were labeled (micellar casein) and frozen. 23 1.2. Human breast milk Human breast milk was obtained from healthy mothers who agreed to donate breast milk samples in amounts that did not jeopardize the baby's nutritional supplement (10-60 mL). Samples were obtained up to 70 days postpartum by breast pump or occasionally by hand and were processed within 2 h after collection. When not explicitly indicated otherwise, all steps of the process were performed as described in 1.1. above. 7.2 mL of an aqueous solution of 200 mM CaCl 2 (mmol / L) was added to 715 mL of whole milk to obtain a final concentration of 2 mM (mmol / L) Ca 2+ concentration. After centrifugation for the cream separation (as described in 1.1, above), 26.6 g of cream were recovered as the top layer and 713 g of skim milk were separated. The separation of sweet whey / casein is obtained by enzymatic treatment of lean milk inducing casein coagulation. Thin milk (200 g) was heated at 35 ° C and then 100 μl of rennet was added immediately under moderate magnetic stirring. After 1 min, the mixture was incubated for 50 min at 35 ° C in a water bath, poured into bottles for centrifugation and subsequently centrifuged at 13600 g for 30 min (as described in 1.1.) In order to separate sweet whey of non-soluble rennet casein.

199 ml of supernatant (ie, sweet whey) were fractionated in 10 x 1.3 ml aliquots (Eppendorf) and 40 ml plastic tubes, which were labeled (Sweet Dairy Whey) 24 and frozen by immersion in liquid nitrogen and stored in a plastic bag at -20 ° C. The rennet casein (2 g) was dispersed in 98 ml of a 2 mM CaCl 2 aqueous solution (mmol / L), said CaCl 2 solution being supplemented with 0.9% by weight NaCl. The resulting mixture was centrifuged; the supernatant (99 mL) was separated and then partitioned into reservoirs, which were labeled (rennet casein wash) and frozen in liquid nitrogen. Recovered 1.15 g rennet casein was dispersed in a 2 mM CaCl 2 aqueous solution (mmol / L), said CaCl 2 solution having been supplemented with 0.9% by weight NaCl in order to obtain a volume of 100 mL. The resulting mixture was then divided into reservoirs, which were labeled (rennet casein) and frozen. The separation of lactic acid whey from casein is obtained by chemical acidification of lean milk inducing casein coagulation. 200 ml of skim milk at 25 ° C were acidified by the addition of an aqueous solution of HCl (32%) from pH 6.6 to pH 4.6 under moderate magnetic stirring. After 1 min of stirring, the mixture was incubated for 60 min at 25øC, poured into bottles for centrifugation and subsequently centrifuged at 13600 g for 30 min (as described in Example 1.1) in order to separate lactic acid whey from casein soluble acid.

The 195 ml of supernatant (ie, acid whey) were fractionated into 10 x 1.3 ml aliquots (Eppendorf) and 40 ml plastic tubes, which were labeled (Lactose 25 acid) and frozen by immersion in nitrogen liquid and stored in a plastic bag at 20 ° C. Acid casein (4.9 g) was dispersed in 95 mL of a 20 mM (mmol / L) aqueous sodium acetate solution having pH 4.6, and the mixture was centrifuged. The supernatant (96 mL) was divided into reservoirs, which were labeled (acidic casein wash) and frozen in liquid nitrogen.

The 2.9 g recovered from washed acid casein were dispersed in 95 mL of water. The pH of this mixture was adjusted to 6.2 by the addition of NaOH. Subsequently, the volume was adjusted to 100 mL. The mixture was then divided into reservoirs, which were labeled (Casein Acid) and frozen.

In order to separate soluble proteins (whey) and washed non-soluble proteins (micellar casein), the following steps were performed: The skim milk was ultra-centrifuged as described in 1.1.

The 198 mL supernatant was fractionated in 10 x 1.3 mL aliquots (Eppendorf) and 40 mL plastic tubes, which were labeled (soluble whey protein) and frozen by immersion in liquid nitrogen and stored in a plastic at minus 20 ° C. Micellar casein (0.5 g) was dispersed in 32 mL of a 2 mM aqueous CaCl 2 solution (mmol / L), said CaCl 2 solution being supplemented with 0.9 wt% NaCl and ultra-centrifuged (as described in Example 1.1). The supernatant thus obtained (32.8 mL) was divided into reservoirs, which were labeled (micellar casein wash) and frozen in liquid nitrogen.

The 0.4 g recovered of washed micellar casein were dispersed in a 2 mM CaCl 2 aqueous solution (mmol / L), said CaCl 2 solution having been supplemented with 0.9% by weight NaCl, to obtain a volume of 33 mL. The resulting mixture was divided into reservoirs, which were labeled (micellar casein) and frozen. 1.3. Buffalo milk Pure fresh buffalo milk was obtained in the Lahore region of Pakistan and supplied refrigerated to a nearby laboratory where it was lyophilized. The freezing temperature was about -20 ° C to about -25 ° C and the drying temperature was 40 ° C. The vacuum pressure was maintained below 2 Torr, with a final pressure of 0.2 Torr. The dry powder was sealed in aluminum-coated plastic bags, shipped and then stored at room temperature. When not explicitly stated otherwise, all steps of the process were performed as described in Example 1.1. The lyophilized buffalo milk was reconstituted at 13% total solids (TS) by dissolving 70 g of the powder in 468 mL of H2 O, and adding 5.4 mL of a 200 mM CaCl2 aqueous solution (200 mmol / L) . 543 ml of reconstituted buffalo milk having a final concentration of 2 mM Ca 2+ were obtained. After centrifugation for cream separation, as shown in Example 1.1, 46 g of cream were recovered as an overcoat and 485 ml of skim milk were separated. Separation of sweet whey from casein was obtained by enzymatic treatment of lean milk inducing casein coagulation. 145 mL of skim milk at 35 ° C was heated, then 73 μL of rennet was added immediately under moderate magnetic stirring. After 1 min the mixture was incubated for 50 min at 35 ° C in a water bath, poured into bottles for centrifugation and subsequently centrifuged for 30 min at 13600 g (as described in Example 1.1) in order to separate sweet whey of non-soluble rennet casein.

The 130 ml of supernatant (ie, sweet whey) were fractionated in 10 x 1.3 ml aliquots (Eppendorf) and 40 ml plastic tubes, which were labeled (Sweet Dairy Whey) and frozen by soaking in nitrogen and stored in a plastic bag at -20 ° C. The rennet casein (14 g) was dispersed in 140 ml of a 2 mM aqueous CaCl 2 solution (mmol / L), said CaCl 2 solution being supplemented with 0.9% by weight NaCl, and centrifuged (as presented in Example 1.1). The supernatant thus obtained (130 mL) was divided into reservoirs, which were labeled (rennet casein) and frozen in liquid nitrogen.

The 13 g recovered casein from the washed rennet were dispersed in an aqueous solution of 2 mM CaCl 2 (mmol / L), said CaCl 2 solution having been supplemented with 0.9% by weight NaCl, to obtain a volume of 145 mL. The resulting mixture was then divided into reservoirs, which were labeled (rennet casein) and frozen. The separation of whey acid / casein is obtained by chemical acidification of lean milk inducing casein coagulation. 145 ml of skim milk at 25 ° C were acidified by the addition of 0.6 ml of aqueous HCl solution (32%) of pH 6.74 to pH 4.6 under moderate magnetic stirring. After 1 min of stirring the mixture was incubated for 60 min at 25øC, poured into bottles for centrifugation and subsequently centrifuged for 30 min at 13600 g (as described in Example 1.1) in order to separate lactic acid whey from casein soluble acid.

The 129 ml supernatant was fractionated into 10 x 1.3 ml aliquots (Eppendorf) and 40 ml plastic tubes, which were labeled (acidic whey) and frozen by immersion in liquid nitrogen and stored in a plastic bag at minus 20 ° C. Acid casein (13 g) was dispersed in 130 ml of 20 mM aqueous sodium acetate solution (pH 4.6), and the mixture was centrifuged (see Example 1.1). The supernatant (129 mL) was divided into reservoirs, which were labeled (acid casein wash) and frozen in liquid nitrogen.

The 12 g recovered from washed acid casein were dispersed in water and the pH was adjusted to 6.7 by addition of NaOH. Subsequently, the volume was adjusted to 145 mL. The mixture was then divided into reservoirs, which were labeled (Casein Acid) and frozen.

In order to separate out soluble proteins and washed non-soluble proteins (micellar casein), the following steps were performed: 145 ml of lean buffalo milk were ultra-centrifuged (as described in Example 1.1) to separate whey from non-soluble micellar casein . The 132 mL supernatant was fractionated in 10 x 1.3 mL aliquots (Eppendorf) and 40 mL plastic tubes, which were labeled (soluble whey protein) and frozen by immersion in liquid nitrogen and stored in a plastic at minus 20 ° C. The gelatinous micellar casein (11.4 g) was dispersed in 133 ml of a 2 mM aqueous CaCl 2 solution (mmol / L), said CaCl 2 solution being supplemented with 0.9% by weight NaCl. The mixture was ultra-centrifuged (see 1.1, above). The supernatant thus obtained (125 mL) was divided into reservoirs, which were labeled (micellar casein wash) and frozen in liquid nitrogen.

The 11 g of washed micellar casein were dispersed in an aqueous solution of 2 mM CaCl 2 (mmol / L), said CaCl 2 solution being supplemented with 0.9% by weight NaCl, to give a volume of 145 ml. The resulting mixture was then divided into reservoirs, which were labeled (micellar casein) and frozen. 30

Example 2

Sub-fractionation of soluble whey protein from Bovine Milk 15 ml of soluble whey protein, obtained as described in Example 1, were thawed for 20 min in a water bath at 37øC, mixed by vortexing and centrifuged for 1 min at 13,000 rpm in an Eppendorf 5415 centrifuge. After filtration on a 0.45 μm Millipore filter (306 / GSWP04700.GS), 10 ml of this preparation was injected onto an HR16x50 column filled with 100 ml of Source 15 RPC TN 17-0727-02 (polystyrene-divinylbenzene), which was linked to a FPLC system controlled by a UNICORN station (Amersham Pharmacia Biotech).

The chromatographic conditions were:

Buffer A: 0.1% TFA in water (2000 ml of mil filtered water in a Millipore system of 0.45 Âμm, plus 2 ml of TFA (Sigma 91699, 100 ml)):

Buffer B: 80% acetonitrile, 0.85% TFA (400 mL of miliq water filtered in a Millipore system of 0.45 Âμm, plus 1600 mL of acetonitrile, degassed in an ultrasonic bath for 15 minutes, and finally, supplemented with 1.7 mL of TFA). The column was equilibrated with 20% buffer B. Then, after a column volume (CV), the sample was injected, buffer B raised to 75% at 15 CV and to 100% at 2.5 CV. At the end, the gradient decreased to 20% buffer B at 0.4 CV. The flow rate was set at 3 mL / min. 96 18 mL fractions were collected in plastic tubes. The fractions were maintained at -20 ° C. After analysis by HIC-HPLC, the 96 collected tubes were pooled in 14 fractions by HPLC profile resemblance and concentrated by evaporation prior to lyophilization for subsequent screening. Table 1 shows 14 sub-fractions of whey protein by a percentage vol. of buffer B or a range in acetonitrile, in which the sub-fractions are eluted. 32

Table 1

Soluble whey protein sub-fractions defined by hydrophobic interaction chromatography% Initial Buffer B% Final Buffer B% Initial Acetonitrile% Final Acetonitrile 1 20 26 16 20.8 2 26 35 20.8 28 3 35 43 28 34.4 4 43 44.5 34.4 35.6 5 44.5 47 35.6 37.6 6 47 48.5 37.6 38.8 7 48.5 50 38.8 40 8 50 51 , 5 40 41.2 9 51.5 52.5 41.2 42 10 52.5 54 42 43.2 11 54 58 43.2 46.4 12 58 60 46.4 48 13 60 70 48 56 14 70 100 (80% / 19.15% / 0.85%; v / v) Column: Source 15 RPC Amersham (matrix: Polystyrene / divinylbenzene) ), column volume (CV = 100 mL) Gradient: starting from 20% Buffer B, the sample was injected after 1 column volume (CV), then the gradient increased to 75% B at 15 CV, followed by 2 CV to reach 100% of Buffer B 33

Example 3

(A permanent endothelial cell line derived from the vein of a normal human umbilical cord; ATCC CRL1730; M. Miralpeix, M. Camacho et al., Brit. J. Pharmacol , 121 (1997), 171-180) were plated in 96-well plates (RPMI 1640 + 10% FCS) and treated with 100 nM (nmol / L) phorbol 12-myristate 13-acetate for 6 h at 37 ° C to induce the COX-2 isoenzyme. 50 μΜ (pmol / L) arachidonic acid were then added and cells were incubated in the presence of test compounds or mixtures for 30 min at 37 ° C. Prostaglandin-E2 production was measured by radioimmunoassay (RIA) and radioactivity determined with a scintillation counter (Topcount, Packard) using a liquid scintillation cocktail (Packard).

The results obtained are expressed as a percentage of control values and as a percent inhibition of control values obtained in the presence of the test compounds. The control values are obtained by performing the above steps, wherein, however, instead of ... (it seems that something is still missing - please indicate). The concentration causing half maximal inhibition of control values (IC50 value) and Hill coefficients (nH) were determined for reference compounds (cycloheximide) by non-linear regression analysis of their inhibition curves. These parameters were obtained by interpolation of the Hill equation. The IC 50 values obtained for the reference compounds were within the accepted limits of historical means obtained ± 0.5 log units.

All COX-2 screening experiments were performed once. In the following, inhibition values less than 10% were not considered and were indicated as " 0 ". 2.2. Human milk fraction COX-2 screening Human lean milk has a significant inhibition of COX-2 (25%; Table 2). When casein is removed by acidification or rennet (giving acid whey and sweet whey, respectively), 51% and 58% inhibition of COX-2 were achieved. Thus, casein removal greatly improved COX-2 inhibition. In addition, single caseins (acid, sweet, micellar) exhibit no inhibitory effect on COX-2. The soluble whey of human milk performed similarly to the starting product (i.e., lean human milk). 35

Table 2

Results of the COX-2 rater with human milk fractions. TS, total solids Human Milk Thin TS code (mg / mL) dilution to 50 g / mL Estimated protein (mg / mL) COX-2 inhibition (%) Lean human milk (comparative) -04 10 25 human milk H3 80 6,25E-04 6 51 (comparative) HM H4 sweet whey 80 6,25E-04 6 58 HM soluble whey (comparative) H5 80 6,25E-04 6 26 casein (comparative) H6 10 5,00E-03 10 0 HM sweet casein (comparative) H7 10 5,00E-03 10 0 HM micellar casein (comparative) H8 5 1.00E-02 5 0 2.3 screening of COX-2 of buffalo milk (comparative) lean buffalo milk has an average inhibitory effect (36%; Table 3). Unlike human milk, the acidic removal of caseins leads to a loss of two-thirds of their activity (less than 12%), probably due to a loss of acid-induced activity after acidification, if the pH is kept constant (i.e. in the case of rennet and ultracentrifugation), the activity increases to 44% in both cases. 36

As with human milk, the micellar caseins of buffalo milk showed no activity. Acid and sweet caseins were not assayed because of their lack of solubility.

Table 3

Results of COX -2 tracing with buffalo milk. TS, total solids. (BM) B2 TS (mg / mL) dilution to 50 Âμg / mL estimated (mg / mL) in the sample COX-2 inhibition (%) lean buffalo (BM) B2 B2 5.00E-04 30 36 BM B3 acid whey 70 7.14E-04 6 12 BM B4 sweet whey 70 7,14E-04 6 44 soluble whey of BM B5 70 7,14E-04 6 44 non-soluble BM acid casein BM sweet casein not soluble BM micellar casein B8 25 2,00E-03 25 0 37 2.4 COX-2 screening of cow's milk

High activity of raw and lean cow's milk (52-53%, Table 4) demonstrates that elimination of cream has no effect on COX-2 inhibition. The activity remains constant in soluble whey (53%), decreases slightly in acid whey (45%) and increases in sweet whey (68%). Without being bound to any theory, the decrease in lactic acid whey activity may, as in buffalo milk, be related to the influence of acid, whereas the increase in activity after rennet treatment could be explained by the theory that destabilization of casein releases some " latent " bioactivity, as is sometimes described during " imperfect fractional distillation " leading to yield of greater than 100%. 38

Table 4

Results of COX-2 screening with bovine milk. TS, total solids. (mg / mL) dilution to 50 g / mL estimated in the sample COX-2 inhibition (%) cow's milk CM (comparative) Cl 100 5.00E-04 31 (Comparative) C3 70 7,14E-04 6 45 sweet whey of CM C4 70 7,14E-04 6 68 whey soluble whey (comparative) C2 100 5,00E-04 35 52 CM (comparative) C5 70 7.14E-04 6 53 casein acid CM (comparative) C6 50 1.00E-03 50 42 sweet casein CM (comparative) Cl 50 1.00E-03 50 65 CM micellar casein (comparative) C8 25 2.00E-03 25 0 2.5 COX-2 screening of whey protein preparations (comparative)

Two commercially available whey protein preparations were also screened for COX-2: 39

Lacprodan 80 (MD Foods Ingredients, Viby, Denmark) which contains high amounts of β-lactoglobulin lactosyl forms; WPI 95, a naturally occurring whey protein isolate, which is virtually lactosylated β-lactoglobulin (verified by mass spectrometry). WPI is available at: Davisco Foods International. Inc. 620 North Main, Le Sueur, MN 56058; BIPRO lot JE 251-0-420.

Table 5

COX-2 screening results with whey protein preparations. TS, total solids. (%) Lacprodan 80 LN 100 5.00E-04 80 23 WPI natural milk whey (mg / mL) dilution to 50 g / mL estimated in the sample COX-2 inhibition 95 NW 100 5.00E-04 95 33 cysteine CY 10 5.00E-03 10 0 serum trypsin hydrolyzate DH 100 5.00E-04 80 0 dairy DM 10

As can be seen from Table 4, these compounds also exhibit considerable COX-2 inhibition activity of 40-23% and 33%, respectively. Contrary to this, trypsin proteolysis destroys the COX-2 inhibition activity of whey protein. In addition, the cysteine " anti-oxidant amino acid " has no effect on COX-2 inhibition. 2.5. Comparison of oxidized bovine milk COX-2

Samples of fresh bovine milk (50 mL) were supplemented with peridrol (30% H 2 O 2) to give a final concentration of 0.1 mM, 1 mM, 10 mM, 100 mM (mmol / L) H 2 O in the mixture resulting. All samples were incubated for 4 h at 30 ° C.

Table 6

Results of COX-2 screening with oxidized bovine milk TS, total protein solids Protein Oxidation TS code (mg / mL) dilution to 50 μg / mL (mg / mL) COX-2 inhibition bovine fresh LI 100 5.00E-04 3.1 41 fresh bovine milk H202 0.1 L2 100 5.00E-04 3.1 41 fresh bovine milk h2o2 1 L3 100 5.00E-04 3.1 35 fresh bovine milk bovine milk h2o2 10 L4 100 5.00E-04 3.1 27 fresh h2o2 100 L5 100 5.00E-04 3.1 0 mM 41

Untreated bovine milk and milk treated with 0.1 mM H202 have the same " " inhibition of COX-2 (41%). With h202 at 1 mM, the inhibition decreases slightly (to 35%), further increase to 10 mM reduces COX-2 inhibition to 27%, and with 100 mM H202 - which is, however, far from the physiological conditions no inhibitory activity was observed.

The results indicate that the asset, i. the suppressor component of COX-2 in bovine milk resists H202 at the level of 0.1-10 mM. The decrease in the inhibitory effect corresponds to increased protein oxidation, indicating that the COX-2 inhibitory activity is based on the protein and is strongly related to the natural state of the protein. 2.6. Screening COX-2 of soluble whey protein sub-fractions

The results of COX-2 inhibition by the soluble whey protein sub-fractions are shown in Table 7. Positive values indicate better performance as compared to the control.

Table 7

Results of inhibition of COX II HIC fraction% inhibition of control values 1 21 2 -3 3 -7 42 (continued)

Results of inhibition of COX II fraction HIC% of inhibition of control values 4 7 5 -17 6 -21 7 -55 8 10 9 36 10 41 11 12 12 12 13 10 14 52

Lisbon, November 6, 2008 43

Claims (4)

Use of one or more milk protein fractions exhibiting a cyclooxygenase-2 inhibitory activity for the preparation of a nutritional and / or pharmaceutical composition for the treatment and / or prevention of cyclooxygenase-2 mediated diseases, wherein said milk protein fractions are selected from sweet cow's milk casein, sweet whey from cow's milk and sweet whey from human milk. Use according to claim 1, wherein the nutritional and / or pharmaceutical composition is provided in the oral, topical, parenteral or rectal dosage form. Use according to claim 1 or 2, wherein the cyclooxygenase-2 mediated diseases are selected from the group consisting of cancer and / or precancerous, in particular, cancer and / or precancerous states of the colon, stomach , esophagus, liver, bile, pancreas, head and neck, lung, breast, bladder, female genitals and skin and / or inflammatory diseases, in particular arthritis, rheumatic fever, symptoms associated with influenza or other viral infections, dysmenorrhoea, headache, toothache, degenerative joint disease, and / or Alzheimer's disease. Use according to any one of claims 1 to 3, wherein the pharmaceutical and / or nutritional composition provides a co-treatment during a cancer treatment and / or a treatment of a pre-cancerous condition, in particular during a chemotherapy , a radiation therapy, a biotherapy and / or during a treatment of pain, and / or during a treatment of an inflammatory disease and / or during the treatment of Alzheimer's disease. Use according to any one of claims 1 to 3, wherein the nutritional composition comprises a food material selected from milk, yoghurt, curd, cheese, fermented milks, fermented milk products, ice-cream, cereal products, milk base, infant formulas. Lisbon November 6, 2008 1/4 Fig. 1 2/4 Curd casein Non-soluble protein Casein micellar Fig. 2 3/4 4/4 Fig. 4